Decolorization of synthetic folic acid



Patented Jan. 27, 1953 DECOLORIZATION OF SYNTHETIC FOLIO ACID David I.Weisblat and Arthur R. Hanze, Kalamazoo, Mich., assignors to The UpjohnCompany, Kalamazoo, Mich., a corporation of Michigan No Drawing.Application April 7, 1951, Serial No. 219,901

Claims. 1

This invention relates to the purification of a class of nitrogenheterocycles known as the pterins, and in particular to the purificationof synthetic liver Lactobccillus easel factor or pteroylglutamic acid.

Several methods for the synthesis of L. casei factor, also known asfolic acid and pteroylglutamic acid, have been described by Waller etal., J. Am. Chem. Soc, '70, 19-28 (1948). All known methods ofsynthesizing folic acid, which are dependent upon the condensation ofvarious relatively complex acids and complex amines, result in productscontaining varying proportions of various undesired isomers, oxidationproducts, unreacted intermediates, condensation and polymerizationproducts, hydrolysis and degradation products. Many of these sideproducts are closely related to the biologically active folic acid and,as would be expected, possess similar physical and chemical properties,but for the most part are biologically inactive. Folic acid can beseparated readily from many of its natural contaminants, but it has beendifficult to separate it from those associated pterins which occur inthe mixtures resulting from chemical synthesis.

Numerous methods have been described for the purification of folio acid,such as by adsorption on ion-exchange resins, as described by J. J.Pfiffner et al., J. Am. Chem. Soc. 69, 1476 (1947), by solution inalkali and precipitation of the pteroylglutamio acid therefrom at a pHof about 3.0 to 4.0, as described by Coy W. Waller in U. S. Patent2,466,665, or by solution in a strong acid followed by dilution withwater to effect precipitation of the dissolved folic acid as describedby Coy W. Waller in U. S. Patent 2,474,022.

Although previously known methods for the purification of syntheticfolic acid are satisfactory for the preparation of material containingup to about 80 percent folio acid, they are not very satisfactory forthe commercial production of folic acid for pharmaceutical use, sincethe process must be repeated several times in order to remove theclosely related pterins. A particularly effective method for thepurification of folic acid is described in the copending application byArthur R. Hanze, Serial No, 198,473, filed Novem: ber 30, 1950.According to this method, purification is effected by contacting analkaline solu tion of impure folic acid with a finely divided solidparticulate oxide, hydroxide or carbonate of a metal of group II of theperiodic table having an atomic number between 12 and 56, bothinclusive, for example, calcium hydroxide and magnesium hydroxide,separating the solid therefrom, and recovering the folic acid from theclarified alkaline solution. In this manner it is possible to obtainmaterial containing more than 90 percent folic acid from materialcontaining about percent folic acid.

Although material containing more than percent folic acid is of sumcientbiological activity for therapeutic use, in many instances material ofthis purity contains a slight amount of highly colored pigment whichcauses it to be rejected for failure to comply With the U. S. P.requirements as to color.

The principal object of the present invention is to provide a processfor the purification of folic acid containing highly colored impuritiesto a product that complies with the U. S. P. requirements as to color.

It has been found, quite unexpectedly, that the highly colored pigmentwhich is present in such substantially pure synthetic folic acidpreparations, can be oxidized by hydrogen peroxide to a form which canbe conveniently and rapidly separated from the folic acid. Folic acidwould be expected to react to form an amine oxide; however, under theconditions of the process of the present invention, the hydrogenperoxide reacts with or affects only the unwanted pigment.

In accordance with the process of our invention, the highly coloredpigment contaminating the substantially pure synthetic folic acid can beseparated or destroyed by dissolving the crude folic acid in diluteaqueous alkali, adding a small amount of hydrogen peroxide, allowing thesolution to stand for a period between approximately 15 minutes andapproximately 1 hour, separating the insoluble precipitate which forms,and recovering folic acid from the clarified alkaline solution. Amongthe bases that can be employed in the preparation of the alkalinesolution of folic acid are lithium hydroxide, potassium hydroxide,sodium hydroxide, calcium hydroxide, barium hydroxide, sodium carbonate,potassium carbonate, and the like. From 90 to 98 percent or more of thefolic acid originally present in the solution can be recovered inpurified form by merely heating the clarified solution to about 90degrees centigrade or higher, adjusting the pH of the solution toapproximately 3.0 and allowing the folic acid to crystallize therefrom.The crystals of folio acid which separate upon acidification at anelevated temperature have a more desirable crystalline form than thoseobtained when the acidification is conducted at or about roomtemperature.

As the excess base must be neutralized in order to precipitate thepurified folic acid, only an amount of alkali sufficient to dissolve thecrude folio acid and its associated genetic impurities should be used,about three molecular equivalents of base per mole of folic acid beingmost satisfactory.

Solutions containing about 3 to grams of folic acid per liter, andpreferably such as contain approximately 8 grams per liter, can betreated in accordance with the process of this invention. The crudefolic acid can be dissolved directly in the required volume of dilutealkali, or more conveniently in a small volume of a onenormal solutionof alkali and then diluted to a suitable concentration of folio acid forthe subsequent treatment with peroxide. Between about 0.3 and 1.0 gramof hydrogen peroxide per gram of folic acid is satisfactory, about 0.5gram being preferred. Although the process of the present invention isparticularly adapted to the treatment of folic acid having a purity ofat least 65 percent, the use of the process for the treatment ofpreparations of lower purity may require greater proportions of hydrogenperoxide and would accordingly not be as economical.

If desired, the peroxide treatment of the present invention can becarried out in the presence of an inorganic adsorbent such as solidparticulate calcium hydroxide, magnesium carbonate, barium carbonate,magnesium hydroxide, zinc carbonate, and the like, as described in thecopending application of Arthur R. Hanze, Serial No. 198,473, filedNovember 30, 1950 and as disclosed in the examples herein.

The following examples are given by way of illustration only and are notto be construed as limiting.

Example 1 A solution was prepared by dissolving 10 grams of crude folicacid which contained 8.37 grams of pure folio acid in about 65milliliters of onenormal aqueous sodium hydroxide solution and dilutingthe resulting solution to one liter with water.

A mixture of 800 milligrams of powdered calcium hydroxide (slaked lime)and 100 milliliters of the previously prepared solution of folic acidsodium salt was stirred at about 25 degrees centigrade for approximatelyone and one-half hours and filtered through a filter-aid (Celite 545)ad. The filter cake was washed with 25 milliliters of water and thefiltrate and washings were combined and diluted to 250 milliliters withwater. After 10 milliliters of the diluted filtrate was removed forassay purposes, the remaining 240 milliliters was heated to boiling, thepH of the solution adjusted to approximately 3.0 and the solutionallowed to stand in a refrigerator for about twelve hours. Thecrystalline folic acid which formed was separated by filtration anddried to obtain 775 milligrams of material which contained 92 percentfolic acid (88.7 percent recovery) and had a brownish-orange color.

A mixture of 800 milligrams of calcium hydroxide and 100 milliliters ofthe previously prepared solution of folic acid sodium salt was stirredfor one hour. Ten milliliters of 3 percent hydrogen peroxide solutionthen was added while the stirring was continued for 15 minutes, afterwhich an additional 10 milliliters of 3 percent aqueous hydrogenperoxide solution was added and stirring was continued for an additional15 minutes, whereupon approximately 30 milligrams of powdered calciumhydroxide was added and the stirring was continued for 10 minutes. Themixture was then filtered through a filter-aid (Celite 545) pad and thefilter cake washed with 25 milliliters of water. The filtrates were thencombined and diluted with water to a volume of 250 milliliters. After 10milliliters of solution had been removed for assay purposes, theremaining 240 milliliters of clarified solution was heated toapproximately degrees centigrade and the pH adjusted to approximately3.0. After the solution had been cooled for about 6 hours, thecrystalline material was separated by filtration and dried to obtain 745milligrams of material which assayed 95.3 percent folic acid (88.3percent recovery) and which was of satisfactory light yellow color andconformed to the U. S. P. specifications in this respect.

Example 2 A solution of crude folic acid was prepared by dissolving 10grams of material which contained 8.71 grams of folic acid in 65milliliters of onenormal sodium hydroxide and diluting with water to avolume of 1000 milliliters.

A mixture of 100 milliliters of the above solution of folic acid sodiumsalt and 800 milligrams of powdered calcium hydroxide was stirred atroom temperature for 1% hours, filtered through a filter-aid (Celite545) pad, the filter cake washed with 25 milliliters of water and thefiltrate and washings combined. To the combined filtrate was then added10 milliliters of 3 percent aqueous hydrogen peroxide solution and themixture stirred for 30 minutes, whereupon a brown solid settled out.Approximately 25 milligrams of calcium hydroxide was then added and thestirring continued for 45 minutes, the solution filtered through afilter-aid (Celite 545) pad, the filter cake washed with 25 millilitersof water, the filtrates combined, and diluted with water to a volume of250 milliliters. After 10 milliliters of the diluted solution had beenremoved for assay purposes, the remaining 240 milliliters was heated toapproximately 100 degrees oentigrade and the pH adjusted toapproximately 3.0 by the addition of one-normal hydrochloric acid. Theresulting mixture was allowed to stand for about 12 hours in arefrigerator, filtered and the precipitate dried to obtain 770milligrams of material which assayed 95.8 percent folic acid (88.2percent return) and had a light yellow color that conformed to U. S. P.specifications in this respect.

Eatample 3 To 100 milliliters of the solution of folic acid sodium saltprepared in Example 2, which contained a total of 0.871 gram of folicacid, was added 10 milliliters of 3 percent aqueous hydrogen peroxideand the solution was stirred for approximately 30 minutes, whereupon abrown solid settled out. The solution was then filtered through a pad offilter-acid (Celite 545), the filter cake washed with 10 milliliters ofwater and the filtrates and washings were combined and diluted withwater to a volume of 250 milliliters. The solution was heated, acidifiedto a pH of approximately 3.0 and the folic acid recovered as in Example2. The color and purity of the folic acid were improved by thistreatment.

Example 4 A mixture of 100 milliliters of the solution of folic acidsodium salt prepared in Example 2, which contained a total of 0.871 gramof folic acid, and 800 milligrams of powdered magnesium hydroxide wasstirred at room temperature for 1 hours, filtered through a filter-aid(Celite 545) pad, the filter cake washed with 25 milliliters of waterand the filtrate and washings combined. To the combined filtrate wasthen added milliliters of 3 percent aqueous hydrogen peroxide solutionand the mixture stirred for 30 minutes, whereupon a brown solid settledout. Approximately 25 milligrams of magnesium hydroxide was then addedand the stirring continued for 45 minutes, the solution filtered througha filteraid (Celite 545) pad, the filter cake Washed with 25 millilitersof water, the filtrates combined, and diluted with water to a volume of250 milliliters. After 10 milliliters of the diluted solution had beenremoved for assay purposes, the remaining 240 milliliters was heated toapproximately 100 degrees centigrade and the pH adjusted toapproximately 3.0 by the addition of one-normal hydrochloric acid. Theresulting mixture was allowed to stand for about 12 hours in arefrigerator, filtered and the precipitate dried to obtain 766milligrams of material which assayed 95 percent folic acid (88 percentreturn) and had a light yellow color that conformed to U. S. P.specification in this respect,

Inasmuch as the foregoing specification comprises preferred embodimentsof the invention, it is to be understood that the invention is notlimited thereto and that variations and modifications can be made inconventional manner by those skilled in the art without departing fromthe scope of this invention or the claims hereinafter.

We claim:

1. A process for the purification of synthetic folic acid containinghighly colored impurities comprising contacting an aqueous alkalinesolution of the folio acid with hydrogen peroxide, whereby the highlycolored impurities are oxidized and precipitated, removing theprecipitate thus formed, and recovering folic acid of improved color andpurity from the alkaline solution by acidification.

2. A process for the purification of a synthetic folic acid ofsubstantial purity containing highly colored impurities, which comprisescontacting an aqueous alkaline solution of the folio acid with about 0.3to 1.0 part of hydrogen peroxide per part of folic acid for a periodbetween approximately 15 minutes and approximately one hour, whereby thehighly colored impurities are oxidized and precipitated, removing theprecipitate and recovering folic acid of improved color and purity fromthe alkaline solution by acidification.

3. A process for the purification of a synthetic folic acid ofsubstantial purity containing highly colored impurities, which comprisesdissolving the folic acid in a minimal proportion of an aqueous alkalinesolution and diluting the solution to a concentration within the rangeof approximately 4 to approximately 8 grams of folic acid per liter ofsolution, contacting the alkaline solution with an amount of hydrogenperoxide equivalent to about 50 percent by weight of the folic acidcontent of the solution for a period between approximately 15 minutesand approximately one hour, whereby the highly colored impurities areoxidized and precipitated, separating the precipitate from the alkalinesolution and recovering folio acid of improved color and purity from theclarified alkaline solution by acidification.

4. A process for the purification of a synthetic folic acid having apurity of at least percent but containing highly colored impurities,which comprises dissolving the folic acid in an amount of one-normalaqueous sodium hydroxide solution equivalent to approximately 3 moles ofsodium hydroxide per mole of folio acid, diluting the resulting alkalinesolution with water to a concentration equivalent to approximately 8grams of folic acid per liter of solution, contacting the alkalinesolution with approximately 8 grams of solid finely divided calciumhydroxide and about 4 grams of hydrogen peroxide per liter of solutionfor a period of approximately 1 hours, separating the precipitatedimpurities, solid calcium hydroxide and pterins adsorbed thereon, andrecovering folic acid of improved color and purity from the clarifiedalkaline solution by acidification of the solution to a pH ofapproximately 3.0.

5. A process for the purification of a synthetic folic acid having apurity of at least 65 percent but containing highly colored impurities,which comprises dissolving the folio acid in an amount of one-normalaqueous sodium hydroxide solution equivalent to approximately 3 moles ofsodium hydroxide per mole of folic acid, diluting the resulting alkalinesolution with water to a concentration equivalent to approximately 8grams of folic acid per liter of solution, contacting the alkalinesolution with approximately 8 grams of solid finely divided magnesiumhydroxide and about 4 grams of hydrogen peroxide per liter of solutionfor a period of approximately 1 hours, separating the precipitatedimpurities, solid mag nesium hydroxide and pterins adsorbed therefrom,and recovering folic acid of improved color and purity from theclarified alkaline solution by acidification of the solution to a pH ofapproximately 3.0.

DAVID I. WEISBLAT. ARTHUR R. HANZE.

No references cited.

1. A PROCESS FOR THE PURIFICATION OF SYNTHETIC FOLIC ACID CONTAININGHIGHLY COLORED IMPURITIES COMPRISING CONTACTING AN AQUEOUS ALKALINESOLUTION OF THE FOLIC ACID WITH HYDROGEN PEROXIDE, WHEREBY THE HIGHLYCOLORED IMPURITIES ARE OXIDIZED AND PRECIPITATED, REMOVING THEPRECIPITATE THUS FORMED, AND RECOVERING FOLIC ACID OF IMPROVED COLOR ANDPURITY FROM THE ALKALINE SOLUTION BY ACIDIFICATION.